Oral tolerance is defined as a temporary loss of systemic immunological responsiveness to a specific soluble antigen after ingestion of that same antigen. Results from a number of studies suggest that ligation of CTLA-4 by B7 in the absence of IL-12 may play a primary role in the induction of suppressed Th1 cell immune responses (e.g., decreased levels of IgG2a, cellular proliferation and IFN-g production). Since CD40-CD40L interaction is known to stimulate IL-12 production by APC's, the absence of this signal may lead to decreased levels of this cytokine and subsequent suppression of Th1 cell responses. To determine this, the ability of stimulatory anti-CD40 mAb administered in vivo (with and without anti-CTLA-4 mAb) to enhance IL-12 secretion and prevent the suppression of Th1 immune responses in mice will be investigated. Further studies will be performed to determine if B7-CTLA-4 interaction and low levels of IL-12 are required for the continued suppression (i.e., maintenance) of Th1 immune responses; these experiments will involve the in vitro addition of anti-CD40 mAb and anti-CTLA-4 mAb to cultures of previously tolerized murine cells. In addition, IL-10 has been shown to regulate IL-12 levels by inhibiting it's production. Therefore, the ability of in vivo and in vitro administration of anti-lL-10 mAb, in conjunction with anti-CTLA-4 mAb, to prevent and maintain suppressed IL-12 and Th1 immune responses in tolerized mice will also be investigated. ELISA and PCR techniques will be used to determine IL-12 concentrations and the levels of Th1 (IFN-g) and Th2 (IL-4)-specific cytokines. IgGAM as well as Th1 (IgG2a) and Th2 (IgG1)-specific antibody isotypes will also be assessed by ELISA. Cellular proliferation responses will be determined using a BrdU colorimetric immunoassay. Elucidation of the mechanisms underlying the generation of oral tolerance is critical to the understanding and subsequent treatment of autoimmune diseases such as multiple sclerosis, uveitis and rheumatoid arthritis. It is expected that results from these experiments will enhance our understanding of these mechanisms, thus facilitating progress in the management of autoimmune disease. [unreadable] [unreadable]